The present invention relates to methods of screening pigs to determine the presence or absence of osteopontin (OPN) alleles associated with increased litter size, to the use of such methods in predicting litter size in pigs and to kits for carrying out such methods.
Meat production and animal breeding efficiencies could be improved if it were possible to increase animal litter sizes. The same output of livestock could be derived from fewer parent animals, thus providing decreased production costs. In addition, animal breeding organisations would benefit from the potential to screen more offspring for those with improved genetics. However, litter size is very difficult to select for conventionally as it is limited to one sex and is heavily influenced by non-genetic factors (heritability, a measure of the fraction of the phenotypic variation that is due to genetic differences is approximately 0.1 for litter size in the pig).
One approach to improving litter size might be to introduce beneficial genes into production lines from breeds which have significantly higher litter sizes. However, quantitative genetics suggests that complex traits such as litter size are controlled by a large number of genes each having a small effect on the trait. If this is true, genetic progress through selection of complex traits is likely to be very slow. An alternative view is that, although many genes are involved in complex traits, a few of the genes involved (major genes) have large effects on the trait. If this alternative view is true, then genetic progress of such traits could be rapid, provided that it is possible to identify and select for beneficial alleles of relevant major genes. Since the advent of genome mapping, it has become possible to identify genes affecting quantitative traits (quantitative trait loci, QTL) by looking for associations between the trait and molecular markers distributed evenly across the genome of animals for which maps are available. Importantly, for selection purposes, the heritability of such marker phenotypes is 1.0.
The Chinese Meishan breed of pig is known to produce about 4 extra piglets per litter than the most prolific European breeds. Genes for prolificacy (litter size) from this breed would be of great value in programmes aimed at increasing the litter size of commercial Western pig breeds. Indeed a genetic marker associated with the oestrogen receptor gene (ESR) of the Meishan has been shown to have beneficial effects on litter size and is described in WO92/18651.
The Booroola Merino breed of sheep is extremely prolific. Litter sizes of three or more are common. The significantly increased prolificacy of this breed has been shown to be due to the action of a single gene, FECB (for review see G W Montgomery, et al, Endocrine Reviews, 13: 309-328 (1992)). Genetic mapping using human DNA markers has shown that the human version of FECB is located on chromosome 4 (G W Montgomery, et al, Nature Genetics, 4: 410-114 (1993)) and is closely associated with the gene encoding secreted phosphoprotein-1 (SPP-1), also known as osteopontin (OPN), 2ar, bone sialoprotein-1, 44 kDa bone phosphoprotein and tumour secreted phosphoprotein. Comparative mapping (H Ellegren, et al, Genomics, 17: 599-603 (1993) has shown that human chromosome 4 and porcine chromosome 8 are highly similar (syntenic). The porcine SPP-1 gene is also located on chromosome 8.
More recently, it has been shown that a FECB-linked marker in cattle does not act as a marker for increased litter size in herds selected for increased ovulation rate (Blattman et al, Mid-West Animal Science Meeting, 18: 43 (1995)).
However, we have surprisingly found that, in pigs, certain DNA markers for OPN are associated with litter size, and thus can be used to select for pigs with a greater chance of producing increased litter size and to select against pigs which have alleles indicating smaller litter sizes. As used herein xe2x80x9cincreased litter sizexe2x80x9d means a significant increase in litter size above the mean of a given population.
It is interesting to note that there is an apparent break point in the chromosome synteny around OPN between sheep, cattle and man on the one hand and mouse and pig on the other (Montgomery et al, J. Reproduction and Fertility supplement, 49:113-121 (1995)). This suggests that the structure of the chromosome may be altered in this region, between animals having large litters (mouse and pig) and those with small litters (man, sheep and cow), such that the effect of the major gene for fecundity is modified. Possible explanations include the expression of the major gene may have been increased or decreased by being brought into a more transcriptionally active or inactive region; the major gene may have been brought directly under the control of an altered promoter element; the position of the major gene relative to OPN may have been changed such that OPN becomes a more useable marker in assessing litter size potential in the pig than in sheep or cattle.
Thus, in a first aspect, the present invention provides a method for screening pigs to determine those more likely to produce larger litters, and/or those less likely to produce larger litters, which method comprises the steps:
(i) obtaining a sample of genomic DNA from a pig; and
(ii) analysing the genomic DNA obtained in (i) to determine which OPN allele(s) is/are present.
Suitably, step (ii), namely the determination of OPN alleles, is carried out by looking for particular DNA markers linked either directly or indirectly to OPN.
Association between genetic markers and genes responsible for a particular trait can be disrupted by genetic recombination. Thus, the closer the physical distance between the marker and the gene in question, the less likely it is that recombination will separate them. It is also possible to establish linkage between specific alleles of alternative DNA markers and alleles of DNA markers known to be associated with a particular gene (e.g. the OPN gene discussed herein), which have previously been shown to be associated with a particular trait. Thus, in the present situation, taking the OPN gene, it would be possible, at least in the short term, to select for pigs likely to produce larger litters, or alternatively against pigs likely to produce smaller litters, indirectly, by selecting for certain alleles of an OPN associated marker through the selection of specific alleles of alternative chromosome 8 markers. Examples of such markers known to be linked to OPN on porcine chromosome 8 include Sw61, Sw1085, Sw194, Sw16, SW790 and SO178, which markers are all microsatellites.
In a further embodiment of the invention a number of such markers are used. For example, pairs of markers might be utilised to bracket the major gene to reduce any possible effects of recombination. Examples of such combinations of markers include SO178 and SW61 and SO178 and SW790.
Since the effect may be related to the difference in gene orders of pigs (and mice) and sheep (and humans and cattle), this suggests that the most useful second marker will be in the non-homologous (non-syntenic) region of pig chromosome 8. An example of a suitable combination of markers known to bracket this region would be OPN and SO178. However, the skilled man will appreciate that other useful markers could routinely be identified.
A particular genetic marker associated with OPN is a microsatellite. These are simple sequence repeats of 4, 3 or, more usually, 2 nucleotides, which occur essentially at random around the genome at approximately every 50,000 bases (about 60,000 microsatellites per haploid genome). Stuttering of DNA polymerase during replication and unequal crossing-over during recombination are thought to result in the loss or gain of repeat units. This means that microsatellites are usually polymorphic and can have several repeat length alleles.
An example of a microsatellite associated with a given gene is (CA)n, resulting in possible repeat unit length alleles, e.g. (CA)2, (CA)9, (CA)10, (CA)11 and (CA)12.
Using primers capable of hybridising (for example, under stringent conditions) to regions flanking the microsatellite associated with the given gene, in combination with standard PCR techniques, PCR products of differing lengths can be generated, the length being dependent on the particular repeat unit length allele of the microsatellite.
Analysing the association of such PCR products using the microsatellite associated with the OPN gene with litter size has allowed marker length alleles associated with increased, and decreased, litter size to be determined in pigs.
Suitable primer pairs which will hybridise to flanking regions of such microsatellites include those having the following sequence:
GCTAGTTAATGACATTGTACATAA; or (SEQ ID NO 1)
CCAATCCTATTCACGAAAAAGC; and (SEQ ID NO 2)
GTGTCATGAGGTTTTTTCCACTGC; or (SEQ ID NO 3)
CAACCCACTTGCTCCCAC (SEQ ID NO 4).
In particular, repeat unit length alleles for the above-noted microsatellite marker, designated 132 and 136, have been found to be associated with increased litter size in pigs. In addition, the repeat unit length allele, designated 112, has been found to be associated with reduced litter size in pigs.
In fact, the allele associated with increased litter size predominantly derived from a European parent stock. This is contrary to expectations since, as discussed above, the Meishan has four extra piglets per litter than either Landrace or Duroc, and it might have been expected that beneficial markers would have been associated with genes inherited from the Meishan parent stock.
In a second aspect the present invention provides a method of screening pigs to determine those more likely to produce larger litters, and/or those less likely to produce larger litters, which method comprises the steps:
(i) obtaining a sample of genomic DNA from a pig;
(ii) hybridising the genomic DNA from (i) with one or more suitable primers;
(iii) carrying out one or more PCR cycles using the hybridised nucleic acid from (ii); and
(iv) analysing the length of the PCR product obtained in (iii).
Suitably, the methods of the present invention are carried out using reagents and instructions presented in the form of a kit.
Thus, in a third aspect, the present invention provides a kit for screening pigs to determine those more likely to produce larger litters, and/or those less likely to produce larger litters, which comprises one or more reagents or materials capable of identifying OPN alleles in a sample of pig genomic DNA.
A preferred kit of the invention will comprise reagents or materials capable of identifying alleles associated with DNA markers linked to the OPN gene, eg the microsatellite marker. Such a kit -would most preferably comprise one or more DNA primers optionally together with standard PCR reagents.
Finally, the skilled person will realise that the methods and kits described herein can be used in conjunction with other already described methods and kits to screen pigs to determine those more likely to produce larger litters (or those less likely to). An example of such other methods and kits are those described in WO92/18651. It would, of course, be possible to produce combined kits which could be used to screen pig DNA using both methods.
In WO-A-9218651 and U.S. Ser. No. 08/312312 there are disclosed methods for determining which pigs are more likely to produce larger litter sizes based on a linkage with the ESR gene. The skilled man will appreciate, therefore, that the screening methods of the present invention can be combined with the earlier disclosed ESR screening methods to provide a yet more powerful tool for such determinations. Thus, in a further aspect, the present invention provides a method for screening pigs to determine those more likely to produce larger litters, and/or those less likely to produce larger litters, which method comprises the steps:
(i) obtaining a sample of genomic DNA from a pig;
(ii) analysing the genomic DNA obtained in step (i) to determine which OPN allele(s) is/are present; and
(iii) analysing the genomic DNA obtained in step (i) to determine which alele(s) of at least one other gene linked to litter size in pigs is/are present.
In one preferred embodiment of this aspect of the invention the at least one other gene is the ESR gene, as described in WO-A-9218651 and U.S. Ser. No. 08/312312.
In a final aspect the present invention provides a kit for screening pigs to dtermine those more likely to produce larger litters, and/or those less likely to produce larger litters, which comprises one or more reagents or materials capable of identifying OPN alleles in a sample of pig genomic DNA, together with one or more reagents or materials capable of identifying alleles of at least one other gene linked to litter size in pigs in a sample of pig genomic DNA.
Preferred features of each aspect of the invention are applicable to each other aspect mutatis mutandis.
The invention will now be described with reference to the following examples, which should in no way be construed as limiting the invention.